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DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 劉?睿(Je-Ruei Liu) | |
dc.contributor.author | Zong-Yuan Wu | en |
dc.contributor.author | 吳宗原 | zh_TW |
dc.date.accessioned | 2021-06-15T02:39:24Z | - |
dc.date.available | 2014-08-14 | |
dc.date.copyright | 2009-08-14 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-08-12 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44088 | - |
dc.description.abstract | 聚木糖(xylan)為植物半纖維素中含量最為豐富的多醣,其水解為許多產業上所必需。以單功能酵素進行聚木糖的水解反應較為緩慢,而聚木糖酶(xylanase)與聚木糖修飾取代基支鏈之水解酵素具有加乘效果,故能加速聚木糖的水解。因此,尋找雙功能或多功能的聚木糖水解酵素有其必要性。本研究室近日自瘤胃真菌Neocallimastix patriciarum S20之cDNA基因庫中分離到雙功能纖維分解酵素XynS20E之cDNA,其具有2,016 bp之開放讀碼區(open reading frame),可轉譯成全長671個胺基酸,分子量72.4 kDa之蛋白質。
本研究的目的為利用大腸桿菌表達XynS20E之全長型與截斷型酵素(分為不含醣苷水解酶結構區或不含醣苷水解酶與錨定結構區等二種截斷型酵素),並分析酵素之基本特性。將全長型與截斷型xynS20E基因選殖於大腸桿菌表達載體後,再以大腸桿菌大量表達帶有組胺酸標籤之重組蛋白質,並利用固定化金屬 離子親和性層析(immobilized metal ion affinity chromatography)進行酵素之純化。純化之XynS20E-6xHis,再以反應曲面法(response surface methodology)分析其最適作用條件。結果於pH 8.2、58℃時,XynS20E-6xHis之比活性最高(795.57 U/mg of total protein)。在熱安定性方面,XynS20E-6xHis於80℃培養2小時後,其活性仍維持在起始活性的70%以上。在受質專一性方面,XynS20E-6xHis可與數種受質(如燕麥聚木糖、樺木聚木糖、乙醯聚木糖、7-乙酰氧基-4-甲基香豆素及1, 2, 3, 4-tetraacetyl-xylopyranose等)反應並釋出乙酸。在酵素動力學分析方面,以樺木聚木糖為受質,XynS20E-6xHis於在最適反應條件之Km與Vmax分別為0.167 ± 0.024 mg/mL與0.086 ± 0.005 µmol/min ∙ mg。比較全長型與截斷型重組XynS20E之活性,全長型酵素的活性較不含醣苷水解酶結構區或不含醣苷水解酶與錨定結構區等二種截斷型酵素分別高出1.16及1.31倍。 本研究結果顯示純化之XynS20E-6xHis具有極佳的熱安定與耐鹼性,因此,XynS20E在未來的產業應用上具有極大的潛力。 | zh_TW |
dc.description.provenance | Made available in DSpace on 2021-06-15T02:39:24Z (GMT). No. of bitstreams: 1 ntu-98-R96626017-1.pdf: 7906722 bytes, checksum: 23652e5d528db47ee85d23981d27c88e (MD5) Previous issue date: 2009 | en |
dc.description.tableofcontents | 中文摘要 1
英文摘要 2 第一章、序言 4 第二章、文獻探討 5 一、聚木糖酶與乙醯聚木糖酯酶 5 (一)聚木糖之主要結構及水解酵素 5 (二)乙醯聚木糖酯酶 5 (三)聚木糖酶與乙醯聚木糖酯酶之交互作用 7 二、瘤胃真菌 7 (一)絕對厭氧真菌之發現 7 (二)絕對厭氧真菌之分離源 8 (三)瘤胃真菌生活史 8 (四)瘤胃真菌之分類地位 8 (五)絕對厭氧真菌在瘤胃中扮演之角色 10 第三章、材料與方法 16 一、實驗材料 16 (一)乙醯聚木糖酯酶基因 16 (二)載體 16 (三)菌種 16 (四)培養基 16 (五)試藥 16 (六)實驗儀器及器材 18 二、實驗方法 19 (一)XynS20E之胺基酸序列與結構區域分析 19 (二)帶有組胺酸標籤(His-tag)之乙醯聚木糖酯酶表達質體之建構 20 (三)重組蛋白質之大量表現 21 (四)重組乙醯聚木糖酯酶之純化 21 (五)聚丙烯醯胺膠體電泳 22 (六)蛋白質定量 23 (七)酵素之活性測試 24 (八)XynS20E-6xHis乙醯聚木糖酯酶最適作用之pH值與溫度 25 (九)XynS20E-6xHis乙醯聚木糖酯酶之熱安定性 26 (十)XynS20E-6xHis乙醯聚木糖酯酶之.受質專一性及酵素動力學 26 第四章、結果與討論 39 一、XynS20E之酵素特性 39 二、XynS20E之胺基酸序列與結構區域分析 39 三、XynS20E重組酵素全長型及截斷型之大量表達與純化 41 四、XynS20E-6xHis乙醯聚木糖酯酶之最適反應溫度與pH值 42 五、重組XynS20E乙醯聚木糖酯酶之結構域活性 44 六、XynS20E-6xHis乙醯聚木糖酯酶之熱安定性 44 七、XynS20E-6xHis乙醯聚木糖酯酶之受質專一性與酵素動力學 45 第五章、結論 62 參考文獻 63 作者小傳 72 | |
dc.language.iso | zh-TW | |
dc.title | 瘤胃真菌乙醯聚木糖酯酶XynS20E重組酵素之純化與特性分析 | zh_TW |
dc.title | Purification and characterization of acetylxylan esterase XynS20E from Neocallimastix patriciarum | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 陳明汝(Ming-Ju Chen) | |
dc.contributor.oralexamcommittee | 林慶文(Chin-Wen Lin),王政騰(Zheng-Teng Wang),陳小玲(Hsiao-Ling Chen) | |
dc.subject.keyword | 瘤胃真菌,乙醯聚木糖酯酶, | zh_TW |
dc.subject.keyword | rumen fungi,acetylxylan esterase, | en |
dc.relation.page | 72 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2009-08-12 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 動物科學技術學研究所 | zh_TW |
顯示於系所單位: | 動物科學技術學系 |
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